Many important physical characteristics of samples are best determined by optical imaging. Conventional optical microscopy, however, is limited in its ability to resolve very small objects by the wavelength of the light, and conventional "far-field" optical microscopes reached their limit of resolution more than a century ago. A variety of techniques have since been used to extend the resolution of optical microscopy, including: confocal microscopy; near-field methods using probes with a limiting aperture; photon tunneling; and aperture less methods that monitor light scattering from a sharp tip. Each of these techniques has significant limitations. Existing techniques with resolutions much better than the diffraction limit in air typically suffer from low light levels, fragile probes, and difficulty in distinguishing the optical information from other physical parameters, such as distance from the sample.
Conventional optical microscopes using objective lenses illuminate the specimen with an external light source and use lenses in the far-field to gather and focus the light. The far-field corresponds to a specimen-lens distance of many optical wavelengths. In 1877, Abbe published a fundamental formula: ##EQU1##
According to Abbe's formula, conventional far-field optical microscopes using objective lenses cannot resolve features with a separation smaller than d, where lambda is the wavelength of light and NA is the numerical aperture of the objective lens. The numerical aperture is determined by: EQU NA=nsin.THETA. 2!
Where n is the refractive index of the lens, and theta is the half-angle of the cone of illumination.
In the 1880's, oil immersion objectives reached a numerical aperture of 1.4 allowing light microscopes to resolve two points separated by approximately 0.2 microns (approximately lambda/3 for visible wavelengths). With the exception of very unusual (and most often toxic) immersion fluids, and the use of ultraviolet light, this remains the limit for conventional optical microscopes today.
Confocal Microscopes
The principle of laser scanning confocal microscopy (LSCM) was first taught by Minsky (U.S. Pat. No. 3,013,467) in the 1950's. In this well-known technique, light from a point source illuminates a very small region of a sample, and a point detector detects light from that small area. By limiting the spatial dimension of the detector, images with resolution better than the classical diffraction limit may be obtained. An image of the sample object forms one point at a time by synchronously scanning the light source and the detector, in the same way that a scanned television image is formed one pixel at a time. Confocal microscopy has many advantages over standard optical microscopy. For example, confocal microscopy allows for optical sectioning (i.e., depth discrimination) of translucent specimens and provides images of the surface topography of reflective opaque specimens. In addition, a confocal microscope has a horizontal resolution Ip to 1.4 times that of a conventional microscope, and can screen out fogginess normally observed with standard microscopes used on living specimens.
The prior art also includes confocal microscopes using an incoherent light source and an imaging detector to form a complete image in real-time without having to synchronously scan the light source or detector. This full-field confocal microscope uses a spinning disc with an array of pinholes, called the Nipkow disc, as a spatial filter in place of the point detector. The common attributes of confocal microscopes are the ability to image axial "slices" of a specimen, and slightly improved lateral resolution compared to conventional microscopes using objective lenses.
Near-Field Microscopes
In 1928 Synge suggested that optical microscopy could overcome the diffraction limit of light by abandoning the far-field and instead working in the near-field (E. A. Synge, "A Suggested Method for Extending Microscopic Resolution into the Ultra-Microscopic Region", Phil. Mag. 6 (1928) p. 356-362). The near-field exists in close proximity to the specimen, less than one optical wavelength. Using a tiny aperture and placing that aperture in the near-field of the specimen, optical microscopy can achieve significantly greater resolving power.
A number of different implementations of Synge's idea have since been developed. Pohl (U.S. Pat. No. 4,604,520) suggested coating the tip of a prism-like crystal; a sharply pointed optically transparent body is covered with an opaque layer into which an opening is formed at the apex of the body, the opening having a diameter small compared to the wavelength of the light used. A group at Cornell "taffy-pulled" glass micropipettes down to sub-wavelength diameters and defined the aperture by metallic overcoats. Betzig (U.S. Pat. No. 5,272,330) improved upon the Cornell pulled micropipette by replacing the glass micropipette with a fiber optic cable. Using the fiber optic cable Betzig increased transmission efficiency (optical throughput) by three or foul orders of magnitude.
While Betzig improved efficiency, a fundamental problem remains. Although light will propagate efficiently down a fiber optic cable of standard diameter, the light becomes "choked off" when the diameter is reduced beyond a certain dimension. Light propagates in a waveguide-like fashion in a fiber optic cable of standard diameter, but when the diameter of the inner core is reduced, the propagating mode gives way to an evanescent mode. In the evanescent mode the optical energy is no longer truly propagating and is no longer confined to the fiber optic core, but rather a portion of the energy dissipates in the metallic overcoat or escapes by backreflection up into non-propagating modes of the fiber optic cable. The longer the distance light must travel in this evanescent mode, the more energy that escapes. A fiber probe with an aperture diameter of 1000 .ANG. has an efficiency of roughly 2.times.10.sup.-4 ; a probe with an aperture of 250 .ANG. has an efficiency of roughly 1.times.10.sup.-6. The efficiency plummets even further for smaller resolutions. Although extremely small apertures can be produced by pulling, the resulting efficiency is so low that virtually no usable light reaches the aperture and the specimen is not illuminated brightly enough to obtain a useful image.
Further improvements to the fiber probe have been proposed, including Islam (U.S. Pat. No. 5,485,536), and Buckland (U.S. Pat. No. 5,410,151). Rather than draw the optical fiber to a thin point and force the light to travel many wavelengths in a non-propagating, evanescent mode, Islam uses a conical tip with a tip length on the order of a few wavelengths. Buckland proposes the use of mulitmode fiber rather than single mode fiber and control of the rate of the taper of the probe tip to improve efficiency. However, the principle of operation is the same: confine the light with an opaque metallic overcoat and force it through a sub-wavelength aperture, or pinhole.
Near-field fiber probe microscopes have additional limitations. In controlling the tip-sample distance, fiber probe microscopes are limited by the fragile nature of the fiber and require very sensitive control of tip-sample distance or very smooth sample surfaces to avoid damage. The requirement to place the surface to be inspected at a distance from the membrane that is approximately equal to the diameter of the aperture implies the limitation that only objects can be inspected that have a surface flatness significantly better than an optical wavelength. In addition, this kind of near-field microscope is particularly difficult to implement for opaque samples where the fiber probe and the conventional collection optics must share the limited space above the sample surface.
Photon Tunneling Microscopes
The basis of operation of the scanning tunneling optical microscope (STOM), also known under the name photon scanning tunneling microscope (PSTM), is the sample-modulated tunneling of internally reflected photons to a sharply pointed optically transparent tip (Ferrell et al., U.S. Pat. No. 5,018,865). The source of the photons is the evanescent field produced by the total internal reflection (TIR) of a light beam from the sample surface, which essentially provides an exponentially decaying waveform normal to the sample surface. Spatial variations in the evanescent field intensity form the basis for imaging. Photons tunneling from the total internal reflection surface to the tip are guided to a suitable detector that converts the light flux to an electrical signal.
These microscopes use a collimated (not focused) beam traveling incident on the surface at an angle greater than the critical angle. They are limited to transparent samples, which must be optically coupled to the prism using an index-matching gel or oil. The beam illuminates a large area of the sample (typically about 1 mm.sup.2). A tapered fiber tip perturbs the evanescent field and some of the light from the sample "leaks" out and gets collected by the fiber tip. A photodetector connected to the fiber monitors the collected light.
This technique is limited by the requirement that samples must be transparent; also, a strong background signal due to the large spot size produces stray light by scattering from dirt and defects in the prism and sample. Mixing between optical properties and surface topography can produce images of very small features, but the demonstrated resolution is comparable to conventional far-field optical microscopes (200 nm, or lambda/3 for visible light). The data depends also on whether moisture becomes trapped between the tip and the surface, complicating the internal reflection. The tips used are fragile and there is no force feedback to control tip-sample forces.
Akamine (U.S. Pat. No. 5,489,774) proposed a photon tunneling probe that replaces the tapered fiber with photosensitive cantilever. A photosensitive region on the lower surface of the lever collects light generated by local disruption of the evanescent field or frustrated total internal reflection (FTR). The sharp tip on the lever is used to locally perturb the evanescent field of the sample surface. The cantilever is patterned with wires to connect the photosensitive region to a photodiode current measurement circuit. In addition, a cantilever deflection sensor 16 provides sensitive force feedback. Akamine has the same limitations as fiber-probe photon tunneling microscopes and also is very sensitive to electrical noise, since the photodetector has an active area much larger than the tip dimensions.
A "full-field" photon tunneling microscope was proposed by J. M. in 1987 Guerra (U.S. Pat. No. 4,681,451). In place of a probe having a sharp tip the microscope uses a thin film transducer in contact with the sample surface over a large area (determined by the diameter of the front face of the objective lens). The microscope measures surface topography using totally internally reflected light. Annular illumination provides high lateral resolution (approximately lambda/4), and the exponential decay of the evanescent wave gives a vertical resolution of 1 nm. Unfortunately samples must be very flat because the maximum vertical range (depth of field) is only lambda/2, and the transducer has a large contact area.
In addition, the microscope proposed by Guerra cannot separate height variations from a change in refractive index. A height variation changes the separation between the transducer and the sample, and a change in refractive index alters the decay length of the evanescent wave. Both effects produce the same optical contrast. Thus it is sometimes necessary to make a replica of the sample using optically isotropic material (having a constant refractive index) to avoid confusion between height and refractive index variations.
Combined Atomic Force and Scanning Energy Microscopes
A variety of combined atomic force (AFM) and scanning energy microscopes have been proposed. When using a near-field technique for optical imaging, the additional information provided by an AFM capability can allow the optical characteristics of the sample to be distinguished from probe height effects. Since a scanning energy microscope typically must have the capability to scan an optical probe over a sample, the incorporation of AFM capability may appear fairly straightforward. Most optical probes are not suited for use as AFM probes, however, and many of the proposed combined microscopes suffer from very low light levels and alignment difficulties.
Hansma (U.S. Pat. No. 5,581,082) describes a Combined Scanning Probe and Scanning Energy Microscope comprising an Atomic Force Microscope (AFM) integrated with a Laser Scanning Confocal Microscope (LSCM). Rather than scan the AFM probe or the laser beam of the LSCM separately, the invention uses a sample scanner to provide AFM and LSCM images simultaneously and in registration.
The focusing element in Hansma is a conventional microscope objective and the lateral optical resolution is the same as conventional LSCM's. The probe of the invention is a conventional AFM tip providing height information. Simultaneous optical and height imaging requires precise alignment to position the LSCM laser spot on the AFM probe tip. The Hansma microscope is designed primarily for transparent samples. On opaque reflecting samples the lateral resolution is lower because a long working distance, and thus lower numerical aperture objective, is required. Thus Hansma discloses an LSCM as the means for focusing energy, which is a distinct and separate element from the AFM, the probe, arranged so as to be closely proximate to the sample surface.
The Hansma microscope provides only the limited improvement in resolution of an LSCM, in contrast to near-field scanning optical microscopes, which can have resolutions much better than the diffraction limit. Also, since Hansma uses an AFM probe that is separate from the optical components, the design has significant potential alignment problems.
An aperture less near-field optical microscope was disclosed by Wickramasinghe (U.S. Pat. No. 5,602,820). The microscope uses a standard AFM tip, with a sharpness on the order of an atomic dimension. A conventional object source to a diffraction light source to a diffraction-limited spot that illuminates the end of the AFM tip. An interferometer monitors scattered light from the tip and the sample. Since there is a strong background signal from the sample and the shank of the AFM tip, it is difficult to detect the scattering from the apex of the tip. Wickramasinghe discloses a dither motion applied to the tip in order to reduce the background signal. The microscope is difficult to implement on opaque, reflecting samples because the objective lens and the AFM cantilever share the space above the sample. Further, the cantilever and the base of the AFM tip obstruct the focused beam.
Solid Immersion Lens Microscopes
A near-field solid immersion optical microscope was proposed by Mansfield and Kino in 1990 (U.S. Pat. No. 5,004,307). The microscope operates in real time using the same principle as the liquid immersion microscope but with the liquid replaced by solid lens of high refractive index material. The microscope is based on a wide-field confocal microscope using an incoherent light source to illuminate a hemispherical solid immersion lens (SIL) placed in direct contact with the sample surface. The light returns to produce an image at the eyepieces or an imaging detector, such as a CCD camera. With a solid lens of refractive index n=2 and 436 nm illumination this microscope could resolve 100 nm lines and spaces, a factor of two improvement in the edge response over a confocal microscope.
The SIL used in the Mansfield and Kino microscope has a spherical surface (top) and a planar (flat, bottom) surface intersecting the center of the sphere. The planar surface must contact the sample over an area at least as large as the desired field of view (typically 50 to 100 .mu.m). High lateral resolution requires that the gap between planar surface and the sample must be a fraction of a wavelength over the entire field of view. Unfortunately, it is not possible to maintain such a small tip-sample gap over the entire contact area, especially on rough samples. Furthermore, sample tilt and surface contamination (particulates, dust and debris) cause variation in the tip-sample gap over the contact area. As a result, current solid immersion optical microscopes cannot achieve high lateral resolution on most sample surfaces.
The Mansfield and Kino microscope also does not provide a force-feedback loop to control forces between the SIL and the sample. A stiff, massive mount (having a low resonance frequency) carries the SIL. As a result, large tip-sample forces can occur, causing SIL or sample damage.
Solid immersion lenses are also well known in optical data storage systems. Corle (U.S. Pat. No. 5,125,750) and Mamin (U.S. Pat. No. 5,497,359), disclose optical. disk systems using a solid immersion lens. The optical assembly includes an objective lens for reading or writing from an optical medium and a solid immersion lens disposed between the objective lens and the medium, with the SIL having a surface closely spaced from the recording medium. The solid immersion lenses used in data storage systems have large bottom surfaces, however, which are suitable for use only on flat surfaces and are therefore not applicable to microscopy applications. The flat bottom surface of the SIL may form an air bearing with the smooth data storage medium to control the size of the gap between the SIL and the disc surface; there is no force feedback loop to control forces on the SIL due to height variations.